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Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast

hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorGUESDON, Gabrielle
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorGOURGUES, Géraldine
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorRIDEAU, Fabien
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorIPOUTCHA, Thomas
hal.structure.identifierAnimal, Santé, Territoires, Risques et Ecosystèmes [UMR ASTRE]
hal.structure.identifierDépartement Systèmes Biologiques [Cirad-BIOS]
dc.contributor.authorMANSO-SILVÁN, Lucía
hal.structure.identifierMICrobiologie de l'ALImentation au Service de la Santé [MICALIS]
dc.contributor.authorJULES, Matthieu
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorSIRAND-PUGNET, Pascal
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorBLANCHARD, Alain
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorLARTIGUE, Carole
dc.date.issued2023-10-16
dc.identifier.issn2161-5063
dc.description.abstractEnThe genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.
dc.description.sponsorshipConstruction et transplantation de génomes semi-synthétiques de Bacillus subtilis, vers le développement de la prochaine génération de châssis bactériens - ANR-18-CE44-0003
dc.language.isoen
dc.publisherAmerican Chemical Society
dc.subject.enCRISPR-Cas9
dc.subject.enCReasPy-Fusion
dc.subject.enMycoplasma spp
dc.subject.enSaccharomyces cerevisiae
dc.subject.encell fusion
dc.subject.engenome editing
dc.subject.engenome fragment capture
dc.subject.engenome transplantation
dc.subject.enin-yeast genome cloning
dc.subject.enmembrane nuclease MnuA
dc.subject.enwhole genome transfer
dc.title.enCombining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast
dc.typeArticle de revue
dc.identifier.doi10.1021/acssynbio.3c00248
dc.subject.halSciences du Vivant [q-bio]
dc.subject.halSciences du Vivant [q-bio]/Biologie animale/Médecine vétérinaire et santé animal
bordeaux.journalACS Synthetic Biology
bordeaux.page3252-3266
bordeaux.volume12
bordeaux.issue11
bordeaux.peerReviewedoui
hal.identifierhal-04522926
hal.version1
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-04522926v1
bordeaux.COinSctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.jtitle=ACS%20Synthetic%20Biology&rft.date=2023-10-16&rft.volume=12&rft.issue=11&rft.spage=3252-3266&rft.epage=3252-3266&rft.eissn=2161-5063&rft.issn=2161-5063&rft.au=GUESDON,%20Gabrielle&GOURGUES,%20G%C3%A9raldine&RIDEAU,%20Fabien&IPOUTCHA,%20Thomas&MANSO-SILV%C3%81N,%20Luc%C3%ADa&rft.genre=article


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